title: Site-specific modification of recombinant proteins: a novel platform for modifying glycoproteins expressed in E. coli.
abstract: The site-specific modification of proteins is expected to be an important capability for the synthesis of bioconjugates in the future. However, the traditional repertoire of reactions available for the direct modification of proteins suffers from lack of specificity, necessitating costly downstream processing to isolate the specific species of interest. (1) Here, we use a well-established, glycan-specific chemistry to PEGylate model glycoproteins, each containing a unique reactive GalNAc attached to a specifically engineered threonine residue. By engineering E. coli to execute the initial steps of human, mucin-type O-glycosylation, we were able to obtain homogeneous site-specifically modified glycoproteins with fully human glycan linkages. Two mucin-based reporters as well as several fusion proteins containing eight-amino-acid GalNAc-T recognition sequences were glycosylated in this engineered glycocompetent strain of E. coli. The use of one sequence in particular, PPPTSGPT, resulted in site-specific glycan occupancy of approximately 69% at the engineered threonine. The GalNAc present on the purified glycoprotein was oxidized by galactose oxidase and then coupled to hydroxylamine functionalized 20 kDa PEG in the presence of aniline. The glycoprotein could be converted to the PEGylated product at approximately 85% yield and >98% purity as determined by comparison to the products of control reactions.
authors.forename: Grant E
authors.initials: GE
authors.lastname: Henderson
authors.forename: Kevin D
authors.initials: KD
authors.lastname: Isett
authors.forename: Tillman U
authors.initials: TU
authors.lastname: Gerngross
mesh_terms: D002236:Carbohydrate Conformation; D004926:Escherichia coli / Q000378:metabolism*; D005692:Galactose Oxidase / Q000737:chemistry / Q000378:metabolism; D006023:Glycoproteins / Q000096:biosynthesis* / Q000737:chemistry*; D006031:Glycosylation; D006801:Humans; D010084:Oxidation-Reduction; D011092:Polyethylene Glycols / Q000737:chemistry / Q000378:metabolism; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry*; D013912:Threonine / Q000737:chemistry / Q000378:metabolism
chemical_list: D006023:Glycoproteins; D011134:Polysaccharides; D011994:Recombinant Proteins; D013912:Threonine; D011092:Polyethylene Glycols; D005692:Galactose Oxidase